The present invention relates to a new motility-promoting protein macromolecule and a process for the isolation of the said motility promoting protein from buffalo serum/plasma: a slaughter house waste.
Forward motility-promoting protein isolated from buffalo blood serum has the potential for the treatment of human infertility (due to low sperm motility) a great social curse in all human races. The product isolated by the present invention has also the potential for improving farm animal breeding with special reference to buffalo a milch animal of great economic importance in many countries including India.
Human infertility is a great social stigma in all human races as it brings personal miseries to the infertile people. It is generally believed that approx. 15% of the couples are infertile. A rough estimation shows that approx. 20 million Indian couples are infertile. It is thus evident that human infertility is a social problem of immense dimension all over the world. One of the main reasons of human male infertility is due to low order of sperm motility with normal cell count or normal sperm motility with lower sperm population in the ejaculated semen. Although several sophisticated Assisted Reproductive Technologies (e.g. IVF in vitro fertilisation, ICSI intracytoplasmic sperm injection) are available to solve the problems of oligospermic and asthenozoospermic patients ( with low sperm motility), these technologies are highly expensive and the success rate is extremely low. To solve the problem of human infertility due to low order of sperm motility, limited studies have been carried out for the isolation of motility promoting proteins from reproductive fluids and blood serum which are known to have sperm forward motility promoting potential(Morita Z. and Chang M. C. J Reprod Fertil 24, 247-254, 1971; Hoskins D. D., Brandt H and Acott T. S. Fed Proc 37, 2534-2542, 1978; Morton B. E., Fraser C. F and Albagli L.Fertil Steril 32, 99-106, 1979; Brown D. V. and Senger P. L. Biol Reprod 23, 271-275, 1980; Mandal M., Banerjee S. and Majumdar G. C. Biol Reprod 41, 983-989, 1989. Acott and Hoskins (Acott T. S. and Hoskins D. D. J Biol Chem 253, 6744-6750, 1978) have partially purified a 37 KDa glycoprotein from bovine seminal plasma/epididymal plasma which is essential for in vitro motility initiation in the immature bovine caput epididymal sperm. Akerlof et al (Akerlof E. Fredricsson B., Gustafson O., Lunell N. O. Nylund L., Rosenborg L., Slotte H and Pousette A., Int J Androl 12, 124-130, 1989) have demonstrated that human blood serum contains a protein (about 200 KDa) that activates forward motility of human ejaculated sperm. They have also made a patent on this human serum motility promoting protein (Akerlof, E. and Pousette, A., Patent No.WO 90 12 032, Oct. 18, 1990). The motility activating protein (approx. mol. Wt 200 KDa) has an isoelectric point of 5.1 and it comprises albumin, apolipoprotein designated as Al and immunoglobulin. The motility activating protein is usable for sperm motility assay and for the treatment of infertility. A pure protein complex of apolipoprotein and immunoglobulin of mol. wt. 180-250 KDa, has been isolated from human serum (U.S. Pat. No. 5,304,464, Apr. 19, 1994; U.S. Pat. No. 5,453,354, Sep. 26, 1995). The macromolecule also comprises albumin in equimolar amount with apolipoprotein Al and immunoglobulin. It has an isoelectric point of 5.1. The product stimulates sperm motility. Antibodies can be raised against this protein complex human infertility caused by sperm immotility can be treated by administering the product. It can be applied for both diagnostic and therapeutic uses. This product is rather expensive as it is derived from human blood serum.
The main object of the present invention is to provide a new motility-promoting protein macromolecule having a molecular mass of 66 kda and a process for the isolation of said promoting protein from buffalo serum/plasma, a slaughter house waste which has potentiality for use in the treatment of human infertility (due to low sperm motility); a social problem of immense dimension in all countries. A major advantage of our isolated product (forward motility stimulating factor) is that as it is a product of slaughter house waste, it is economically more viable as a commercial product than the one from human serum developed earlier (patent Nos. WO 9012032, Oct. 18, 1990; U.S. Pat. No. 5,304,464, Apr. 19, 1994, U.S. Pat. No. 5,453,354, Sep., 26, 1995). No data are available on the motility promoting efficacy of the human serum product on spermatozoa of other species. The product of this invention can activate motility of sperm derived from various species including buffalo, bovine and goat. Another advantage of our product is that it has also the potential for improving the breeding of farm animals with special reference to buffalo; a milch animal of great economic importance in many countries.
The present invention provides a new motility-promoting protein macromolecule and a process for the isolation of the said motility promoting protein from buffalo serum/plasma: a slaughter house waste, which comprises fractionating buffalo serum/plasma, purifying the motility promoting protein from the above fractionated serum/plasma by conventional chromatography and electrophoresis methods.
Further, the present investigation provides a new motility-promoting protein macromolecule and a process for the isolation of the said motility promoting protein from buffalo serum/plasma: a slaughter house waste. The invention comprises (a) purification and characterisation of a novel forward motility-stimulating protein from buffalo blood serum/plasma and (b) the methodologies for the purification of the motility-promoting protein from the buffalo serum/plasma. The motility-promoting factor is a heat stable protein. This invention reports for the first time the purification to apparent homogeneity of a motility-promoting protein from a biological fluid. It activates forward motility of ejaculated sperm of all the species tested (buffalo, bull goat rat hamster and human) indicating that there is no species specificity for its motility activating potential. The molecular mass of the motility promoter as estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis, high performance liquid chromatography and Sephadex G100 gel filtration is 66 KDa. The factor showed high protein specificity and affinity for activating sperm forward motility. The motility promoter at 0.5 xcexcM level showed nearly maximal activity when 60-70% of spermatozoa were forward motility. It acts on the sperm cells with great rapidity to cause instant activation of sperm motility. It is a glycoprotein that binds with high affinity to concanavalin A. It is an acidic protein with isoelectric point of 3.7 (approx.) and its activity is dependent on Mg++. Motility promoting protein has markedly higher efficacy for activating sperm motility than theophylline or bicarbonate or their combination. The sperm motility induced by the factor is greatly stabilized by bicarbonate. Amino acid analysis shows that it is rich in aspartic acid, glutamic acid and leucine. Both the sugar and protein parts of the molecule are essential for its biological activity. Specific receptors of motility promoting protein have been demonstrated on the sperm surface that may mediate the action of the factor on sperm. It is strongly immunogenic. Studies with antisera against the buffalo motility promoting protein (raised in rabbit) show that the antisera cross react with sera derived from goat, bull, cow and human. The data implicate that the motility promoter or motility promoter like proteins are present in the sera of other species. Using the antibody against the motility stimulating factor, the motility promoter has been shown to be present in testis and epididymis although liver is the richest source of it. The product isolated by the present invention is a new motility promoting protein as its characteristics are clearly different from those reported in the literature.
A part of the invention consists of development of the methodologies for the isolation of motility promoting protein from buffalo blood, a slaughter house waste. Motility promoting protein activity is also present in the blood sera of all the species tested including human but buffalo blood is the richest source of this factor(Mandal M., Banerjee S. and Majumder G. C. Biol Reprod 41, 983-989, 1989). The factor can be isolated from blood serum or plasma. Blood serum/plasma was first fractioned with ammonium sulphate. The motility promoter was sedimented with 40-80% saturation of the salt. The salt fraction of motility promoting protein was then subjected to a cation exchange chromatography using a resin such as carboxymethyl cellulose/Sephadex using low ionic acidic buffer (e.g. 10 mM Sodium acetate, pH 5.6). The factor binds to the resin and it can be eluted from the column at higher concentrations of salt such as 0.4 M NaCl. The motility promoting protein can also be purified by anion exchange chromatography using diethylaminoethyl cellulose/Sephadex as the resin and low ionic alkaline buffer (e.g. 10 mM Tris-HCl, pH 8.3) when the motility promoter does not bind to the resin. The partially purified motility promoting protein as obtained after ion exchange chromatography was purified further by molecular sieving chromatography using Sephadex as the insoluble matrix or high performance liquid chromatography column using phosphate buffer of varying pH (6.9 to 7.5) and varying ionic strength (50-100 mM).The motility promoter can as well be purified by adsorption chromatography using insoluble gel matrix (e.g alumina gel). The factor binds to the gel and can be eluted at a relatively high concentration of salt (e.g 1 M Tris HCl, pH 7.4). Chromatofocusing utilising polybuffer exchanger termed as PBE 94 resin(Pharmacia) can as well be used for the purification of motility promoting protein. Motility promoting protein binds to the concanavalin A-Agarose affinity matrix and it can be eluted from the column with xcex1-methyl-D-mannoside. A suitable combination of these procedures lead to approx. 100 to 400 fold purification of the motility promoter from serum/plasma with recovery of approx. 20 to 30%. The partially purified factor can be purified to apparent homogeneity by using native polyacrylamide gel electrophoresis under a variety of electrophoretic conditions including different percentages (7 to 10%) of the polyacrylamide gel. The resulting pure motility promoting protein was approx. 600 fold purified and the recovery of the activity was approx. 20%. Nearly 3 to 4 mg of pure motility promoting protein was obtained per 100 ml of the blood serum/plasma.
This invention is also relating to pharmaceutical preparations comprising a macromolecule of proteinaceous nature which is essentially pure, has a molecular weight of about 66 KDa and activates sperm motility together with any suitable excipient. Examples of suitable excipients are culture media or other salt solutions. The pharmaceutical preparations are prepared according to methods known per se. The pharmaceutical preparations according to the invention have potentiality for the treatment of infertility.